EP0419502B1 - Verfahren zum nachweis von bakterien im harn - Google Patents

Verfahren zum nachweis von bakterien im harn Download PDF

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EP0419502B1
EP0419502B1 EP89906001A EP89906001A EP0419502B1 EP 0419502 B1 EP0419502 B1 EP 0419502B1 EP 89906001 A EP89906001 A EP 89906001A EP 89906001 A EP89906001 A EP 89906001A EP 0419502 B1 EP0419502 B1 EP 0419502B1
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bacteria
sediment
urine
bacterial
container
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EP0419502A1 (de
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Edward S. Hyman
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
    • Y10T436/25125—Digestion or removing interfering materials
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
    • Y10T436/25375—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
    • Y10T436/25375—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
    • Y10T436/255—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.] including use of a solid sorbent, semipermeable membrane, or liquid extraction

Definitions

  • This invention relates to new and improved methods for the detection of bacteria in urine.
  • Urine originates as an ultrafiltrate of plasma and is normally thought to be free of bacteria as it moves from the upper urinary tract to the bladder. Therefore, bladder urine obtained by suprapubic needle aspiration, and perhaps urine properly obtained by a catheter inserted via the urethral meatus, should contain no bacteria. As it is voided from the body however, the urine is frequently contaminated with microorganisms which colonize the distal urethra and/or the perianal area. The most common non-invasive method of obtaining urine samples which attempts to minimize, but rarely completely eliminates contamination involves meticulous swabbing of the urethral meatus and periurethral areas with a bactericidal agent, followed by the collection of a mid-stream "clean-catch" specimen.
  • the specimen should be examined within 1 hour of collection (two hours if the unpreserved urine specimen is refrigerated) to obviate proliferation of bacteria. Although most contaminating microorganisms are avoided by this "clean-catch" method, the collected samples may still contain some contaminants.
  • a small sample of uncentrifuged urine is placed on a slide, heat-fixed, stained, e.g., using Gram stain, and examined with the high dry objective (at 400X magnification) or under oil immersion (at 1000x magnification).
  • a known volume of urine is centrifuged, the sediment resuspended in the residual fluid.
  • a small sample of the sediment is spread on a slide, covered with a cover slip, and examined directly using the high dry objective (i.e. , a wet mount slide preparation).
  • the centrifuged urine sediment is prepared as in (3) above, but the sample is heat-fixed, stained, e.g. , using Gram stain, and examined with the high dry or under oil immersion objective.
  • bacteria may be seen in an aqueous medium under the microscope at as low at 100 diameters or less magnification, but they are usually visualized at 1000 diameters magnification after drying and staining with appropriate dyes. In the past both methods of visualization have been used to examine urine for the presence of bacteria. Using conventional wet mount slide preparations, bacteria usually observed in urine are Gram negative bacilli (rods). Group D streptococci are also seen in classical urinary tract infections (Todd et al., 1984, in Clinical Diagnosis and Management by Laboratory Methods 17th ed., Henry, ed., W.B. Saunders, NY).
  • urinary tract infections are associated with the presence of Gram negative rods such as E. coli, Proteus, the Klebsiella-Enterobacter group, and a lesser number of cocci such as Staphylococcus epidermidis , and the enterococcus.
  • Direct microscopy and culture methods each have disadvantages.
  • bacteria may be seen in urine at only 100 or more diameters magnification, but the size of the image is not the only consideration influencing visibility.
  • the optical density and refractive index of an object be near that of the medium, then it would not be detected by an ordinary light microscopy.
  • Such an object might be seen by appropriate staining with dyes or by specialized lighting such as dark-field illumination, phase illumination, or differential interference.
  • Kunin (1961, New Eng. J. Med. 265: 589) round bacteria cannot be distinguished from other near round particles such as crystals.
  • Kunin (1961, New Eng. J. Med. 265: 589)
  • Any useful culture method requires that the bacteria will grow in the laboratory in the medium selected and in the time allotted. If the medium used is inappropriate for the growth of the particular organisms present, they will not grow. If the time allotted is too short, colonies will not be visible and positive cultures may be mistakenly reported as negative; and if oxygen tension or oxidation potential is either too high or too low, fastidious anaerobic or aerobic organisms may be missed.
  • bacteria in urine might display less than optimal viability.
  • the ionic strength or osmolarity of the urine may be outside the requisite range.
  • the wall of the bacterium may be damaged so that it will require a special medium to grow.
  • the oxidation potential of urine may be too high for growth of a particular bacterial species (e.g.
  • the typical oxidation potential of urine observed by me is about +0,22 to +0,25 Volts, referenced to a saturated calomel electrode. This potential is sufficiently high to inhibit the growth of many bacteria.
  • antibodies against bacteria have been identified in urine and they have been demonstrated to be deposited on bacteria in urine.
  • antibiotics administered to a patient may be excreted in urine in active form at a higher concentration than in other body fluids. The more concentrated antibiotic or antibiotic metabolite is likely to inhibit bacterial growth.
  • Urea (and perhaps other metabolites) present in all urine inhibits bacterial growth. Any one or a combination of these factors may inhibit or diminish bacterial growth in vitro.
  • US-A-4,673,637 relates to a method of urine specimen preparation comprising intense centrifugation and a lipid wash in order to mitigate or prevent loss of bacteria-containing sediment prior to examination; it also establishes that bacteria are present in the urine of patients suffering from rheumatoid arthritis and related autoimmune diseases, hypertension, and other diseases.
  • Rheumatoid arthritis is a chronic systemic inflammatory disease generally regarded as an autoimmune disorder. In addition to inflammation of joints, the disease may cause inflammation and damage to arteries (arteritis), nerves (neuropathy), the sclera of the eye (scleritis), the outer layer of the heart (pericarditis), cardiac muscle (myocarditis), lymph nodes (lymphadenitis), and subcutaneous connective tissue resulting in formation of rheumatoid nodules. The disease occurs worldwide.
  • Hypertension is a chronic elevation of blood pressure which is either without apparent cause (i.e. , "essential” hypertension), or which results from a kidney disorder such as partial obstruction of the flow of blood to part or all of the kidney or a kidney infection (i.e., secondary hypertension). That secondary hypertension may be associated with a kidney infection (pyelonephritis) has been recognized at least since 1939 (see S. Weiss and F. Parker, Medicine, 19, 221-315, 1939).
  • the present invention presents new and improved methods for detecting and identifying bacteria or bacterial fragments in samples of urine.
  • the present invention proposes improved methods for the preparation of samples for rapid direct microscopic detection, identification, and quantification of bacteria.
  • the present invention provides a method of detecting bacteria or bacterial fragments in a urine sample, comprising:
  • the methods of detection of the present invention can be useful to determine the treatment of other diseases or conditions which include the following: rheumatic fever, systemic lupus erythematosis, scleroderma, dermatomyositis, transient ischemic attacks of the CNS, and glomerulonephritis in various stages.
  • Classic bacteremia or septicemia may cause bacteriuria which is rapidly detectable by this new method.
  • bacteria generally Gram positive cocci
  • renal disorders including renal failure due to congenital polycystic kidneys; renal failure due to otherwise unclassified chronic nephritis"; "brittle" diabetes mellitus; recurring kidney stones; unexplained proteinuria; unexplained edema; and chronic brawny edema ( i.e. , lymphangiitis or elephantiasis not due to non-bacterial parasites).
  • Treatment of these patients with known antibacterial agents sufficient to reduce or eliminate the bacteriuria, benefited these patients as if they had been suffering the now-classic pyelonephritis, but often even more than if they had that entity.
  • bacteriuria has also been demonstrated in patients suffering unexplained severe abdominal pain, proved mesenteric lymphangiitis, unexplained fatigue, and unexplained headaches. Treatment of these patients with antibiotic agents resulted in not only a decrease in the number of bacteria in the urine but also improvement of their clinical symptoms of unexplained etiology. It is believed that such patients suffer a bacterial infestation at some undisclosed body site.
  • the present invention describes rapid and novel methods for determining the presence of live or dead bacteria or bacterial fragments in the urine.
  • the methods described herein are useful as a general diagnostic technique. They can be utilized in the diagnosis of illnesses, and to monitor the effectiveness of antibacterial agents and their dosages in the treatment of the above-listed diseases or conditions. They are particularly useful for the detection of bacteria which are not reliably detected by conventional methods.
  • an amount of an antibiotic effective against the bacteria is administered.
  • the amount necessary is determined by the response of the bacteriuria, an in-vivo test of the agent. Relatively large doses of antibiotic may be necessary. For example, an oral dose of 600 mg. per day of clindamycin is sometimes effective, but doses of 3 to 8.4 grams a day by vein may be necessary to reduce or eliminate the bacteria, the dose being limited by the tolerance or the host for the chemical.
  • the dosages may be adjusted for other routes of administration. It may be necessary to use combinations of antibacterial agents at one time to reduce or eliminate the bacteriuria as monitored by the method herein disclosed, e.g. , clindamycin has been used with gentamycin, tobramycin, piperacillin, one of several cephalosporins, tetracyclines, chloramphenicol, etc.
  • antibiotic therapy has effectively reduced or eliminated the cocci and has alleviated the symptoms, signs, and often the abnormal laboratory findings of the patient.
  • One object of the invention is to detect bacteria in urine that are not detected by conventional methods.
  • Another object of the invention is to detect bacteria rapidly so that the method may be useful as a clinical test.
  • Another object of the invention is to ensure that all bacteria and formed parts of bacteria in a urine sample are collected in the sediment. According to one embodiment of the present invention, this is ensured by adequate centrifugation.
  • Another object of the invention is to ensure retention of the urine sediment on the microscope slide throughout staining.
  • Another object of the invention is to remove conflicting, extraneous, or interfering material from the sediment in the preparation of a slide.
  • the invention provides data useful to alert the physician to the possibility that antibiotic therapy, appropriate for the organisms found in the urine by the new and improved methods of this invention, might improve the patient's condition and to determine a treatment to provide therapeutic relief in cases of rheumatoid arthritis, "essential" hypertension, and other diseases or conditions found by methods described herein to be associated with significant bacteriuria.
  • FIG. 1. is a schematic representation of the steps of an improved method for detecting bacteria in urine samples.
  • Urine samples - preservation According to one embodiment of the present invention, bacteria in urine are detected by direct microscopic examination using the methods presented schematically in Figure 1.
  • Urine samples may be processed either fresh (i.e. , within about one hour and preferably within about 20 minutes after collection) or preserved.
  • Preservation of urine samples is accomplished by addition of about 1% by volume "liquefied phenol" (i.e. , phenol containing sufficient water to render it liquid at room temperature), or by adding 0.1% sodium azide.
  • a trace amount of a proton sensitive vital stain such as tetra-brom phenolphthalein ethyl ester potassium salt and sufficient methanol to obtain a blue-green color. This also stains sediment. Other preservatives have been successfully used.
  • Adjustment of pH If the pH of the urine sample is greater than about 6, small aliquots of acetic acid are added dropwise to adjust the pH to about 6 or lower (preferably 5.0).
  • Centrifugation Preferably, the urine is centrifuged at about 3000-11,000 times gravity for 10-15 minutes. Although slight improvement can be had at a higher centrifugal force, this is an adequate sedimentation force x time for urinalysis according to the present invention. Some novel bacterial forms disclosed herein may require centrifugation at 4000 x g or more. Thus, centrifugation at rates in excess of 4000 x g may be used, if desired. Centrifugal sedimentation forces lower than the preferred range (e.g., less than about 3000 x g) generally are not adequate to sediment some bacteria or bacterial fragments in urine particularly when the urine has a relatively high specific gravity, e.g. , about 1.030.
  • Bacterial cell walls or "bacteria fragments" require a higher sedimentation force. Dead or damaged bacteria may have a lower density than viable bacteria. It is important to apply a strong enough force to sediment all bacteria, especially when the difference between the density of the bacteria and that of the medium is minimal. Generally, it has been found that the preferred centrifugal force of from about 4,000 to 11,000 times gravity for from about 10 to 15 minutes is adequate.
  • Step 3 (a) is an optional step utilized where the urine sediment from Step 3 contains substances which may interfere with the retention of bacteria on the slide, with the satining of bacteria or bacterial parts, or with visibility of the bacteria in the sediment.
  • interfering substances include soluble salts, glucose, soluble proteins or crystals of uric acid, calcium oxalate, calcium phosphate or the like. The removal of such interfering substances enhances the detection of bacteria and formed elements in the sediment.
  • Step 3(a) preferably is utilized in the following circumstances:
  • Step 3(a) is carried out by twice washing the sediment from Step 3 with a sterile, particle-free aqueous solution, preferably slightly hypertonic to normal serum, each wash step being followed by centrifugation.
  • one preferred wash solution comprises an aqueous solution of from about 0.15 to about 0.25 N sodium chloride to which about 0.005 ml of a wetting agent (e.g., Tween® 80 or Triton® X-100) has been added.
  • a wetting agent e.g., Tween® 80 or Triton® X-100
  • the wash solution may be sterilized and rendered particle-free, for example, by passing it through a filter whose pore size is 0.22 microns or less.
  • fixative e.g. 0.5 ml of the methanolic alcian blue fixative and 15 ml glacial acetic acid to 1 liter of 0.2 M NaCl. If a blue sediment appears with standing, it can be filtered or decanted. If the specimens are to be used for fluorescent staining, however, the alcian blue must be omitted in this step because it absorbs the incident ultraviolet light.
  • the sediment from the performance of Step 2 is dispersed in about 3 ml of the wash solution and is centrifuged at about 4,000 to 11,000 times gravity for about 5 minutes. The supernatant is removed, and the sediment is again dispersed in about 3 ml of the wash solution and again centrifuged.
  • Insoluble proteins are removed by incubation with bacterial or fungal proteases or proteolytic enzymes of animal origin, such as crystalline trypsin and chymotrypsin.
  • Fresh urine is centrifuged as in Step 3 at 11,000 x gravity for 10 to 15 minutes.
  • the supernatant is removed, and a particle-free buffered solution of crystalline trypsin or of a bacterial protease is added to the sediment, preferably with a small amount of particle-free sodium azide solution to prohibit bacterial growth.
  • the tube is incubated, preferably at 37°C for 10 minutes or for a time appropriate for the enzyme at the concentration used.
  • the tube is then centrifuged at about 11,000 x gravity for 5 minutes.
  • Step 3(a) Proteolysis by trypsin may be used to remove previously soluble proteins that have been precipitated by the preservative. If not removed, these proteins may interfere with staining and with visualization of bacteria.
  • This enzyme treatment removes some of the insoluble proteins.
  • Two advantages are noted. First, some of the sediment is removed, but bacteria, degenerated bacteria, bacterial parts, host cells, and casts are usually spared. This provides a means to concentrate important sediment such as bacteria. Second, the staining of some bacteria is changed. Most notable is the ability to detect Gram positive cocci in sediments that contain only Gram negative cocci in their unwashed or washed preparations. Since the Gram positive characteristic is peculiar to the cell wall of these bacteria, it is quite unlikely that proteolytic enzymes would create the conditions for a positive stain (retention of the iodinated crystal violet). Instead, it is likely that such proteolytic enzymes remove a protein from the bacteria, for example an adherent human antibody which had coated the bacterial cell wall and had prevented the gentian violet from penetrating to, or fixing to, the bacteria.
  • Step 4 can be carried out alternately (a) directly from the centrifugation Step 3, or (b) from the wash Step 3(a), or from wet enzyme step 3(b) previously described.
  • the sediment is dispersed in the residual clear fluid (about 0.1 to 0.2 ml. in an ordinary conical centrifuge tube) and the suspension is spread on a clean glass slide.
  • the quantity of this residual liquid and the sediment may be roughly measured by weighing the centrifuge tube with and without the residue, and estimating the specific gravity to be about 1.1.
  • a measured aliquot (0.02 to 0.05 ml.) of the 0.1 ml of sediment can be spread over a 1 or 2 square centimeter area to quantify the bacteria.
  • the slide may now be viewed wet without a cover slip at 100 to 400 x.
  • the slide is then dried slowly by any means suitable for conventional preparation of bacterial slides, e.g. , at about 40-50°C under an airstream (such as an ordinary portable hair dryer).
  • any means suitable for conventional preparation of bacterial slides e.g. , at about 40-50°C under an airstream (such as an ordinary portable hair dryer).
  • a key step of the improved process of this embodiment of the present invention comprises removal of lipids of lipid-soluble substances.
  • the lipids found in urine are in the range of polarity (in a thin layer chromatogram) of the naturally occurring phospholipids such as lecithin, but they do not contain significant phosphorus. Like phospholipids (or other substances in that polarity range), they are surface active agents. Preferably they are removed from the dried sediment by a solvent system such as a mixture of absolute methanol and a halogenated hydrocarbon (e.g., 1,1,1-trichloroethane in absolute methanol at about 30:1 vol/vol ratio) or by methanol alone.
  • a lipid wash solution is prepared by adding about 15 ml. of 1,1,1 -trichloroethane to about 450 ml. of absolute methanol. The sediment is exposed to the lipid solvent system for about 1-30 seconds.
  • Results are improved by fixation at this point.
  • a dilute solution of glutaraldehyde in absolute methanol e.g., 1 ml glutaraldehyde in 450 ml. of absolute methanol
  • alcian blue Merck Index, 10th ed., No. 208
  • the alcian blue solution is prepared by adding 150 mg. of alcian blue and 4.0 ml. of glacial acetic acid to 100 ml. of absolute methanol. Should the dye precipitate, slightly more acid may be added.
  • the dried urine sediment on the slide is exposed to each fixative for about 1-30 seconds.
  • Step 7(a)
  • a slide prepared according to the method of Fig. 1, may be treated following step 6 with a proteolytic enzyme, or the slide may be treated with the enzyme after Step 7's fixation with glutaraldehyde and even after fixation with alcian blue.
  • a solution of enzyme in the appropriate buffer is simply applied to the slide.
  • the slide is incubated at room temperature or at 37°C, washed with sterile saline, fixed again with alcian blue and stained. Steps 8 through 10 are then performed as in FIG. 1.
  • Step 7 may be modified to permit W fluorescence staining by either increasing the glutaraldehyde concentration several fold or the duration of exposure to the fixative ( e.g ., about 5 minutes).
  • the slide may be stained with (a) an ultraviolet fluorescing dye (e.g ., a stabilized solution of acridine orange, or a solution of a chemical like the antibiotic tetracycline), (b) an antibody (e.g ., a fluorescent antibody - preferably monoclonal - to a specific chemical in a particle in the sediment), or any other target specific substance (e.g. ,an enzyme labeled with a fluorescent dye).
  • an ultraviolet fluorescing dye e.g a stabilized solution of acridine orange, or a solution of a chemical like the antibiotic tetracycline
  • an antibody e.g ., a fluorescent antibody - preferably monoclonal - to a specific chemical in a particle in the sediment
  • any other target specific substance e.g. ,an enzyme labeled with a fluorescent dye
  • the urine sediment is treated with other enzymes or antibodies to reveal additional information.
  • Enzymes such as trypsin or other proteases will selectively remove proteins and have been found to uncover bacteria in the sediment that had been masked by protein; i.e. protein which had occluded or otherwise prevented the Gram stain from binding to the bacteria.
  • cocci may be found to be Gram positive after treatment with trypsin.
  • Enzymes may also be used to identify any formed element in microscopy by solubilizing and thus removing the particle. Should a particle be removed by a substrate-specified enzyme, then the nature of the particle is identified (e.g., if a protease dissolves a particle, the particle was a protein).
  • enzymes that may be so employed are amylase (to remove carbohydrate polymers), DNases, RNases, lipases, lecithinases, sphingomyelinases, sialases, neuraminidases, and hyaluronidases. Enzymes may be used to uncover the surface of bacteria so that a specific antibody or enzyme may then bind to that surface. Such antibody or enzyme may be tagged for visualization by binding to it any fluorescent dye,( e.g ., fluorescein, lissamine-rhodamine, etc.).
  • fluorescent dye e.g ., fluorescein, lissamine-rhodamine, etc.
  • polyclonal or monoclonal antibodies which may be utilized are anti-human IgG, IgM, and IgA, to demonstrate the presence of human immunoglobulin or of a chemical on the bacterial surface or on other elements of the sediment. Rinse with water and dry and go to Step 10.
  • Rinse Preferably the slide from any of steps 7, 7(a) or 7(b) is washed with pure methanol to remove residual alcian blue. Alternately, the solution of methanol/1,1,1-trichloroethane (about 30:1 vol:vol) may be used.
  • Non-fluorescent staining A conventional non-fluorescent stain, such as the Gram stain, may be used as may a counterstain such as safranin.
  • the Gram stain has the time-honored advantage of being used in the classification of bacteria.
  • the slide is dried and examined, e.g. , at 1000 diameters without a coverslip.
  • Special optics e.g. , epi-ultraviolet or phase
  • Several other optional steps may also be performed in the method of FIG. 1 for detecting bacteria in urine as follows.
  • stains may be added at any step. This includes "vital” stains which penetrate living and dead cells at different rates and which stain intracellular components differentially. For example, brilliant cresyl blue or trypan blue may be used. These dyes enhance visualization of the structure of formed elements of urine (casts, leucocytes, tubular epithelial cells, etc.) as well as the bacteria or bacterial fragments. Although this step enhances microscopic visualization of formed elements and bacteria at 100 diameters magnification, it is usually not essential.
  • the "vital” stain may be applied at any time before steps 2, 3, 3(a), or 4, or in the preservative.
  • bacteria, bacterial fragments and bacterial antigens demonstrated herein to be associated with rheumatoid arthritis and related diseases, "essential" hypertension, etc. alternatively may be detected in urine samples using antibodies specific for soluble or insoluble antigens produced by such bacteria, these bacteria having been disclosed by this invention.
  • gram positive bacteria include but are not limited to: Streptococcus faecalis; S. faecium; S. mitis; S. mutans; S. mutants ; S. viridans; S. intermedius; S. salivarius ; Staphylococcus epidermidis; Staph. hemolyticus; Staph. hominis; Peptococcus, etc.
  • Monoclonal antibodies offer the advantage that large amounts of monoclonal antibody specific for a single bacterial antigen can easily and inexpensively be produced.
  • the present invention has several basic advantages over the prior art methods, including:
  • the said detection and identification are useful to determine the treatment of the various illnesses by the administration of therapeutically effective dosages of antibiotics specific to the illness.
  • the treatment can be monitored by the detection and identification of bacteria in the urine of the patients being treated.
  • One improvement of the present invention is based on the discovery that urine contains lipids which act as detergents and interfere with adherence of bacteria and bacterial fragments to a slide. Thin layer chromatography of these lipids in a solvent system appropriate for lipids commonly found in human tissues such as phosphatidylcholine (lecithin), phosphatidyl-ethanolamine, phosphatidylserine, and sphingomyelin, reveals that these lipids are in the same range of polarity as human phospholipids. These lipids however, do not contain appreciable phosphorus, and thus they are for the most part not phospholipids.
  • these lipids are allowed to remain with dried urine sediment on a slide, they will cause the sediment to partially or completely release from the slide when an aqueous solution is applied. The bacteria in the sediment would be lost before they could be seen. This is one reason why past methods of direct microscopy have failed to detect significant members of bacteria in the urine of patients with diseases such as rheumatoid arthritis, etc.. and one reason why the teaching with regard to these diseases has been that significant bacteria are not present in the urine. Standard methods of preparing and staining urine specimens for microscopic examination do not provide for the precautionary removal of these lipids.
  • Another improvement of this invention involves the further fixation of bacteria and other urinary sediment onto a glass slide. Whereas de-lipidation of the dried sediment improves adherence of bacteria to a glass slide, much stronger adherence is obtained by the use of certain chemicals which do not interfere with the staining of the adherent sediment. This includes fixation of proteins of the sediment with glutaraldehyde and the fixation of glycoproteins of the sediment with alcian blue.
  • Another improvement involves the removal of crystals and of excesses of water soluble releasing agents such as glucose and soluble proteins by washing the urine sediment.
  • the positive identification of the various types and states of bacteria is also an improvement of this invention. Only live bacteria (and usually non-fastidious) can be detected and identified by the present conventional cultural methods. Culture methods for fastidious bacteria and usually too cumbersome and too expensive for routine use. Dead or fragmented bacteria simply will not grow in any of the culture media.
  • the non-cultural methods of the present invention result in a rapid, yet positive detection and identification of bacteria and bacterial fragments within a time span to accomplish treatment during the same day or even during the same office visit, as opposed to the 3 to 6 days required for culture growth and analysis.
  • the advantages to the practitioner and to the patient are obvious.
  • the treatment is undertaken only after detection and identification of the bacteria (alive or dead, whole or fragmented) has been completed.
  • a proper antibiotic to be administered and the proper dosage is predetermined from the prior analysis.
  • the monitoring of the treatment by the same detection and identification method ensures the eradication of bacteria (live or dead, whole or fragmented) from urine excreted prior to cessation of treatment. It also may indicate the desirability of a change in the treatment, either in the specific antibiotic, the use of a supplemental antibiotic or a variance in the dosage level.
  • the diseases found causally related to microscopic bacteriuria include osteoarthritis, rheumatoid arthritis, and related syndromes such as juvenile rheumatoid arthritis, ankylosing spondylitis, Reiter's disease, palindromic rheumatism, myositis, fibrositis, bursitis, tendonitis, tenosynovitis, the carpal-tunnel syndrome, panniculitis, tempero-mandibular arthritis, sacroiliac arthritis, systemic lupus erythematosis, erythema nodosum, scleroderma, isolated Raynaud's phenomenon, and other rheumatic diseases.
  • Microscopic (coccal) bacteriuria has been associated with rheumatic heart disease, even when there has been no cultural or serologic evidence of the culpable group A beta-hemolytic streptococcus, but there was evidence of smoldering activity of the rheumatic process. It has been found in patients with hypertension, with presumed idiopathic myocarditis, and with mitral valve prolapse, with or without evidence of "microembolism”. Microscopic bacteriuria has been causally associated with various forms of inflammatory bowel disease including Crohn's disease and ulcerative colitis. It is associated with classic migraine and with other kinds of headache, and with Meniere's syndrome.
  • vasculitis It has been found in various forms of vasculitis, not only the vasculitis of rheumatoid arthritis, but also in retinal arteritis and temporal arteritis; and without followup, in Takayasu's disease and Kawasaki's disease. Without strong evidence of a causal relationship, it has also been found in a patient with a dissecting aortic aneurysm, and with the "subclavian steal" syndrome (clotting of a segment of the subclavian artery due unknown cause. The segment is thought to become inflamed first.)
  • Elimination of microscopic bacteriuria has resulted in improvement or even remissions in the nephrotic syndrome in adults and in children; and improvement in glomerulonephritis, in "chronic nephritis" or interstitial nephritis thought to be sterile but progressive. It occurs with otherwise unexplained proteinuria, and many of those patients have been benefited by elimination of the bacteriuria.
  • Microscopic bacteriuria is found in persons with unexplained edema (salt retention), and in classic chronic lymphedema of the legs (e.g., "milk leg”), and those patients are greatly benefited.
  • Elimination of microscopic bacteriuria has also resulted in a remission in a patient suffering from proven retroperitoneal lymphadenitis and in others with unexplained deep abdominal pain that could be due to that disease. Similar results have been had in patients with the symptomatology attributed to a "hiatus hernia". It has been found associated with a crisis in sickle cell anemia * . Isolated ulceris or uveitis has been relieved by relief of the concurrent bacteriuria. Patients with associated lung disorders such as status asthmaticus, chronic obstructive pulmonary disease (COPD), and just subjective dyspnea without COPD have benefited.
  • COPD chronic obstructive pulmonary disease
  • bacteria, dead or alive, detected in urine do not necessarily represent active bacterial infections (colonization) of the urinary tract. Rather it is currently thought that the majority of presently detected bacteria originate from sites of bacterial infections or infestations of other parts of the body, and that the bacteria or bacterial parts are excreted by the kidney.
  • bacteria have been observed sequestered in neutrophils or macrophages voided from the body in the urine. It is postulated that the dead bacteria and "exploded" cocci observed in the urine specimens may have been previously sequestered in neutrophils or macrophages that have been released into the urine and eliminated from the body.
  • the present invention is not to be limited to this or any other mode by which the bacteria may have been introduced into the urine by the kidney.
  • "Damaged” or “exploded” cocci are seen in the company of either large or small Gram positive cocci. Experimentally these forms can be simulated by performing the same staining method on a smear of broken cell walls of staphylococci or streptococci grown in a culture. "Exploded” cocci are thought to be the cell walls of cocci whose contents have been lost when the cell wall (a shell) was opened by host defenses or by previously administered drugs.
  • the method of the present invention has demonstrated a much higher incidence of bacteriuria in hypertensives, perhaps as high as 90%, as compared to that seen with conventional methods for detecting bacteriuria, i.e. , 2-5%. It can be readily shown by staining and microscopy that many of the bacterial forms observed under the microscope using methods of the present invention were not alive at the time the specimen was obtained. For example, some do not contain any nucleic acid, either DNA or RNA, two biochemical components essential to life. Should all of the bacteria in a given specimen be devoid of nucleic acid, then none will grow and the culture of urine is sterile.
  • Urine samples obtained from the hospitalized patients listed below in Table II were initially cultured under conditions favoring the growth of fastidious facultative anaerobic microorganisms. These conditions include culture at 37°C in tryptic soy broth and on blood agar plates placed in an anaerobic chamber containing hydrogen and carbon dioxide. Beta phenyl ethanol was added to at least one medium whenever Gram negative rod-shaped bacteria were also seen in the urine or in a culture medium. Results are illustrated in Table III. The definitions and replicates in Table III are the same as in Table II.
  • Gram positive cocci were recovered from 67 out of 70 samples cultured under such conditions.
  • the most frequently cultured Gram positive organisms include: S. faecalis ; Enterococcus ; Staph. epidermidis ; S. viridans ; S. mitis , etc.
  • Urine samples from a number of the hospitalized patients listed in Tables II and III were cultured by a hospital bacteriology laboratory using conventional urine culture techniques. Results are illustrated in Table IV. ("--" indicates culture was not performed in the hospital laboratory.)
  • the diameter of the field of view in a microscope is 0.0183 cm, there are 3,800 such fields per square centimeter of slide.
  • the field may be photographed.
  • the rectangular marking used to delineate a 35 mm camera field measures 0.0038 x 0.0026 cm.
  • a reticule for the ocular of the microscope may be used which superimposes a square onto the visual field. Using the 1000 diameter optics, this square is 0.0071 cm on a slide, and there are 19,600 such areas per square centimeter of the slide. The reticule is divided into 100 squares for easy counting of the overall square.
  • the average number of the bacteria of numerous samples was determined. Since the area of the smear is known, and the quantity of urine represented per 1 or 2 square centimeters is also known, then the number of each kind of bacteria is obtained by simple multiplication. The remainder of the sediment is used for a count by culture. It is washed into 4 ml of a rich broth (e.g ., trypticase soy broth) and put through serial dilutions to lower the count. One ml aliquots of each dilution are layered onto each of two Blood Agar Plates (BAP).
  • BAP Blood Agar Plates
  • Results presented in Table IV clearly indicate that the present methods may be used as a rapid screening procedure to determine not only the type of bacteria, but also to estimate the bacterial count, and to ensure that a fastidious one is not overgrown and lost.
  • the present microscopic method has important advantages over both the aerobic and anaerobic culture methods used conventionally.

Claims (13)

  1. Verfahren zum Nachweis von Bakterien oder bakteriellen Fragmenten in einer Urinprobe, umfassend:
    a) Zentrifugieren der Probe bei einer relativen Zentrifugalkraft von etwa 3500-11000-facher Schwerkraft zum Sedimentieren von Bakterien und bakteriellen Fragmenten,
    b) Abtrennen des Sedimentes von dem Überstand,
    c) Ausstreichen des Sedimentes auf einer Oberfläche,
    d) Waschen des Sedimentes auf der Oberfläche mit einer Lipid-Lösungsmittelzusammensetzung zur Entfernung jeglicher Lipidkomponenten des Sedimentes vor dem Färben,
    e) Fixieren des gewaschenen Sedimentes mit Glutaraldehyd und Phthalocyaninblau,
    f) Färben des fixierten, gewaschenen Sedimentes, und
    g) mikroskopisches Untersuchen des gewaschenen und gefärbten Sedimentes.
  2. Verfahren nach Anspruch 1, bei dem die Probe bei mehr als 4000 bis 11000-facher Schwerkraft zentrifugiert wird.
  3. Verfahren nach Anspruch 1 oder 2, bei dem die Probe 10 bis 15 Minuten lang zentrifugiert wird.
  4. Verfahren nach einem der Ansprüche 1 bis 3, bei dem die Lipid-Lösungsmittelzusammensetzung eine Mischung von Methanol und Halogenkohlenwasserstoff oder Methanol alleine ist.
  5. Verfahren nach Anspruch 4, bei dem das Lipid-Lösungsmittelsystem eine Mischung von Methanol und 1,1,1-Trichlorethan ist.
  6. Verfahren nach einem der Ansprüche 1 bis 5, bei dem das Sediment ferner einem markierten Antikörper ausgesetzt wird.
  7. Verfahren nach einem der Ansprüche 1 bis 5, bei dem das Sediment ferner einem Fluoreszenzfarbstoff ausgesetzt wird.
  8. Verfahren nach Anspruch 7, bei dem der Fluoreszenzfarbstoff Acridinorange umfaßt.
  9. Verfahren nach einem der Ansprüche 1 bis 8, bei dem das Sediment ferner vor der mikroskopischen Untersuchung einem Enzym ausgesetzt wird.
  10. Verfahren nach Anspruch 9, bei dem das Enzym ausgewählt wird aus der Gruppe bestehend aus Amylasen, DNasen, RNasen, Lipasen, Lecithinasen, Sialasen, Neuraminidasen, Hyaluronidasen, Sphingomyelinasen, bakteriellen Proteasen, Trypsin, und anderen proteolytischen Enzymen.
  11. Verfahren nach Anspruch 10, bei dem das proteolytische Enzym ausgewählt wird aus der Gruppe bestehend aus bakteriellen und fungalen Proteasen, kristallinem Trypsin, und Chymotrypsin.
  12. Verfahren nach einem der Ansprüche 1 bis 11, ferner umfassend das Waschen des Sedimentes mit einer sterilen partikelfreien wässrigen Lösung, die gegenüber normalem Serum leicht hypertonisch ist, um wasserlösliche Substanzen zu entfernen, die entweder mit der Retention von Bakterien auf einem Objektträger oder mit der Sichtbarmachung von Bakterien interferieren.
  13. Kit zur Herstellung von Harnsediment zur Untersuchung, umfassend:
    a) einen Behälter mit einem Lipide entfernenden Lösungsmittel, 0,03 bis 5 Vol.-% 1,1,1-Trichlorethan in absolutem Methanol,
    b) einen Behälter mit einem Fixierungsmittel enthaltend etwa 0,004 bis 0,4 Vol.-% Glutaraldehyd in Methanol,
    c) einen Behälter mit einer angesäuerten methanolischen Lösung von Phthalocyalinblau,
    d) gegebenenfalls Behälter für die Gram-Färbung, einschließlich:
    1) eines Behälters mit einer wässrigen Lösung von Kristallviolett,
    2) eines Behälters mit in wässrigem Kaliumjodid aufgelöstem elementaren Jod,
    3) eines Behälters mit einer Entfärbungslösung,
    4) eines Behälters mit einem Gegenfärbemittel, und
    e) gegebenenfalls einen Behälter mit einer Lösung zum Waschen des Sedimentes, welche aus einer wässrigen Lösung von Natriumchlorid und einer verdünnten Lösung von angesäuertem Phthalocyaninblau besteht.
EP89906001A 1988-06-13 1989-05-17 Verfahren zum nachweis von bakterien im harn Expired - Lifetime EP0419502B1 (de)

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US07/205,959 US4992365A (en) 1984-04-23 1988-06-13 Method of detecting bacteria in urine
US205959 1988-06-13
PCT/US1989/002115 WO1990000201A1 (en) 1988-06-13 1989-05-17 Method of detecting bacteria in urine

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CN112113817B (zh) * 2020-10-09 2023-03-14 嘉兴晶铸生物科技有限公司 一种全自动的脱落细胞制片方法
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WO1990000201A1 (en) 1990-01-11
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CA1340780C (en) 1999-10-05
ATE133206T1 (de) 1996-02-15
EP0419502A4 (en) 1991-11-27
US4992365A (en) 1991-02-12
AU3575089A (en) 1990-01-23
DE68925481T2 (de) 1996-06-13

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